Proteins containing the eukaryotic Cys2His2 zinc finger DNA-binding motif are relatively flexible with respect to their sequence specificity (Wolfe et al., 2000, Ann. Rev. Biophys. Biomol. Struct. 29:183–212; Pabo et al., 2001, Ann. Rev. Biochem. 70:313–340). DNA recognition typically involves a tandem array of two or more zinc fingers. Fingers with a desired sequence specificity can be created by randomizing the residues involved in base-specific DNA recognition sequences and then selecting fingers with the desired specificity from the resulting library or by designing fingers based on understood principles of zinc finger-DNA recognition (Liu et al., J. Biol. Chem., 2002, 277(6):3850–6). For example, fingers with novel specificity have been selected using phage display (Rebar et al., 1996, Meth. Enzymol. 267:129–149) or using a bacterial two-hybrid system (Joung et al., 2000, Proc. Natl. Acad. Sci USA 97:7382–7387). Proteins with a desired sequence specificity are created by making an assembly of fingers that have been selected to have the desired sequence specificity.
Zinc finger proteins have been used successfully in vivo to alter gene expression by attaching repression or activation domains (Beerli et al., 2000, Proc. Natl. Acad. Sci. USA 97:1495–1500; Zhang et al., 2000, J. Biol. Chem. 275:33850–3360; Liu et al., 2001, J. Biol. Chem. 276:11323–11334). Regulation of a gene by zinc fingers has also been made drug-dependent by using two types of accessory domains: 1) a zinc finger-homeodomain fusion protein wherein the association of an activation domain with this protein was rendered drug-dependent (Rivera et al., 1996, Nature Med. 2:1028–1032; Ye et al., 199, Science 283:88–91). This was accomplished by fusing protein domains to the activation and DNA-binding domains that could associate only in the presence of the drug rapamycin; and 2) a zinc finger-steriod receptor fusion protein that binds to DNA only in the presence of synthetic steroid receptor antagonists (Beerli et al., 2000, J. Biol. Chem. 275:32617–32627; Xu et al., 2001, Mol. Ther. 3:262–273).
The tetracycline receptor (TetR) is a component of the tetracycline resistance system (Tn10) in bacteria that represses expression of the tetracycline exporter pump (TetA) (Saenger, 2000, Angew Chem. Int. Ed. Engl. 39:2042–2052). This protein binds to an operator DNA sequence (tetO) as a homodimer in the absence of tetracycline (Tc), but in the presence of the drug it can no longer bind to DNA. Consequently transcription of TetA is no longer repressed. The structure of TetR bound to DNA containing the tetO sequence has been determined (Orth et al., 2000, Nat. Struct. Biol. 7:215–219), as has the structure of TetR with Tc bound (Hinrichs et al., 1994, Science 264:418–420). Examination of these structures suggests that an allosteric mechanism of inactivation of TetR occurs upon binding by Tc (Orth et al., 2000, Nat. Struct. Biol., 7:215–219). TetR is composed of two domains with different functions: the dimerization domain, which has at its core a four helix bundle, and the DNA-binding domains which folds into a helix-tum-helix (HTH) motif. These two domains are rigidly connected by an α-helix. Tc binds in a pocket in the dimerization domain of each monomer, and this results in a conformational change in the dimerization domain that translates down the connecting α-helix, and increases the distance between the helix-turn-helix motif of each monomer by about 3 Å. This is believed to be the reason that the TetR•Tc dimer can no longer bind to DNA containing the tetO sequence.
TetR has been used to form the basis for a drug-regulatable gene regulation system in eukaryotes (Williams et al., 2000, J. Appl. Physiol. 88:1119–1126). TetR is a bacterial protein and consequently, it should not interact with any of the components of the eukaryotic machinery. Moreover, most eukaryotic systems can tolerate Tc (or a similar derivative that binds to TetR) at high concentrations. A specific gene can be rendered drug-inducible simply by attaching a repression or activation domain to TetR, and introducing multiple copies of tetO in front of the gene of interest (Gossen et al., 1995, Science 268:1766–1769). This system has been further augmented by the creation of a modified TetR (rTetR) that cannot bind in the absence of tetracycline, but that binds the operator sequence (tetO) in the presence of the drug (Urlinger et al., 2000, Proc. Natl. Acad. Sci. USA 97:7963–1768). The primary limitation of this system is the necessity to introduce the tetO operator into the promoter of the gene of interest.